Recombinant Anaeromyxobacter sp. ATP synthase subunit b (atpF)

What is the structural organization of ATP synthase in Anaeromyxobacter sp. and how does subunit b fit within this complex?

ATP synthase in Anaeromyxobacter sp., like other bacterial F-type ATP synthases, consists of a membrane-embedded F₀ domain (containing subunits a and c) and a catalytic F₁ domain (α₃β₃γδε). Subunit b forms part of the peripheral stalk that connects these domains. Based on structural studies of related ATP synthases, subunit b serves as a critical component of the peripheral stalk, forming a dimer (b-b') that helps anchor the catalytic α₃β₃ hexamer to the membrane sector . The peripheral stalk, which includes subunit b, functions to prevent rotation of the α₃β₃ hexamer during ATP synthesis or hydrolysis, thereby enabling the enzyme to convert the mechanical energy of rotation into chemical energy stored as ATP.

How does the amino acid sequence of Anaeromyxobacter sp. ATP synthase subunit b compare to other bacterial homologs?

While specific sequence comparison data for Anaeromyxobacter sp. ATP synthase subunit b is limited in the provided research, comparative analysis would typically examine sequence conservation across related bacterial species. The atpF gene encoding subunit b in Anaeromyxobacter sp. likely shares sequence similarities with other deltaproteobacteria, particularly those in the order Myxococcales. In mycobacterial ATP synthases, the peripheral stalk subunits (b-δ:b') play a critical role in smoothening power transmission between the rotary c-ring and the α₃:β₃:γ:ε domain . Similar functions would be expected for the Anaeromyxobacter homolog, though specific sequence adaptations might reflect its unique environmental niche.

What expression systems are most effective for producing recombinant Anaeromyxobacter sp. ATP synthase subunit b?

For optimal expression of recombinant Anaeromyxobacter sp. ATP synthase subunit b, E. coli-based expression systems are commonly employed for bacterial membrane proteins. Based on successful expression of related ATP synthase components, the following methodological approaches are recommended:

• Expression vector selection: pET-based vectors with T7 promoters offer strong, inducible expression

• Host strain optimization: C41(DE3) or C43(DE3) strains are preferred for membrane proteins as they can accommodate potentially toxic membrane proteins

• Induction conditions: Lower temperatures (16-20°C) and reduced IPTG concentrations (0.1-0.5 mM) often improve folding of membrane proteins

• Extraction methods: Specialized detergents like n-dodecyl-β-D-maltoside (DDM) or lauryl maltose neopentyl glycol (LMNG) are effective for solubilizing membrane proteins while preserving native structure

Commercial providers like CUSABIO TECHNOLOGY LLC have successfully produced this protein, suggesting established protocols exist for its expression and purification .

How does the peripheral stalk architecture in Anaeromyxobacter sp. ATP synthase contribute to energy conservation during ATP synthesis?

The peripheral stalk of ATP synthases, including subunit b, plays a crucial role in energy conservation through its function as a stator that prevents unproductive rotation of the α₃β₃ hexamer. In mycobacterial ATP synthases, the peripheral stalk components (b-δ:b') have been shown to smooth transmission of power between the rotary c-ring and the catalytic domain .

Similar to findings in Mycobacterium smegmatis, where movements of peripheral stalk subunit δ visualized different states of the ATP synthase and underscored its function as a transfer element of elastic energy during ATP formation , the Anaeromyxobacter peripheral stalk likely exhibits comparable elastic properties adapted to its metabolic requirements.

What methodological approaches are most effective for studying the interaction between subunit b and other components of the Anaeromyxobacter sp. ATP synthase complex?

To investigate interactions between subunit b and other ATP synthase components in Anaeromyxobacter sp., several complementary methodological approaches are recommended:

• Cryo-electron microscopy (Cryo-EM): This technique has proven valuable for resolving ATP synthase structures in related organisms. For example, cryo-EM has been used to visualize the structure of Mycobacterium smegmatis F₁-ATPase and the F₁F₀-ATP synthase with different nucleotide occupation states . A similar approach could reveal the structural relationships between subunit b and other components in the Anaeromyxobacter complex.

• Cross-linking studies coupled with mass spectrometry: This approach can identify specific interaction sites between subunit b and neighboring proteins. The method involves:

  • Chemical cross-linking of proximate amino acids

  • Enzymatic digestion of cross-linked complexes

  • Mass spectrometric analysis to identify cross-linked peptides

  • Computational modeling to reconstruct interaction interfaces

• Site-directed mutagenesis and functional assays: By introducing specific mutations in predicted interaction regions of subunit b, researchers can assess the functional consequences on ATP synthesis or hydrolysis activities.

• Single-molecule FRET (Förster Resonance Energy Transfer): This technique can measure dynamic changes in protein-protein interactions during the catalytic cycle by monitoring distances between fluorophores attached to subunit b and other components.

What is the role of ATP synthase in the energy metabolism of Anaeromyxobacter sp. in relation to its diverse respiratory capacities?

Anaeromyxobacter species display remarkable respiratory versatility, including the ability to utilize various electron acceptors such as oxygen, nitrate, and metal oxides. ATP synthase plays a central role in coupling this respiratory diversity to energy conservation. The following aspects highlight this relationship:

• Adaptation to fluctuating oxygen levels: Anaeromyxobacter sp. is found in paddy soils where oxygen availability fluctuates . The ATP synthase must function efficiently under both aerobic and anaerobic conditions.

• Integration with nitrogen metabolism: Anaeromyxobacter isolates harbor nitrogen fixation capabilities . This energetically demanding process requires substantial ATP, creating a functional linkage between ATP synthase activity and nitrogen fixation. The ATP synthase must support the high energy demands of nitrogenase (NifHDK complex) activity, which requires approximately 16 ATP molecules per N₂ reduced .

• Potential regulatory interactions: In diazotrophic bacteria like Anaeromyxobacter, ATP synthase activity may be regulated in coordination with nitrogen fixation machinery. The presence of regulatory genes like draT and draG in Anaeromyxobacter strain Red267 suggests sophisticated control mechanisms that likely extend to ATP synthase regulation.

The ATP synthase b subunit, as part of the peripheral stalk, may contain structural adaptations that optimize performance under the diverse respiratory modes employed by Anaeromyxobacter sp.

How can site-directed mutagenesis of the atpF gene be used to investigate the functional significance of conserved residues in Anaeromyxobacter sp. ATP synthase subunit b?

Site-directed mutagenesis of the atpF gene offers a powerful approach to probe structure-function relationships in ATP synthase subunit b. A methodological framework for such investigations includes:

• Identification of target residues:

  • Conserved residues identified through sequence alignment across bacterial ATP synthases

  • Residues predicted to participate in dimer formation

  • Residues at the interface with other ATP synthase components

• Mutagenesis strategy:

  • Alanine scanning to assess the contribution of side chains

  • Charge reversal mutations to test electrostatic interactions

  • Introduction of cysteine residues for cross-linking studies

• Functional assays:

  • ATP synthesis/hydrolysis rates with purified mutant complexes

  • Proton pumping measurements using pH-sensitive fluorescent probes

  • Assessment of complex stability through blue native PAGE

• Structural analysis:

  • Cryo-EM of mutant complexes to detect structural alterations

  • Hydrogen-deuterium exchange mass spectrometry to assess conformational changes

This approach parallels successful mutational studies of ATP synthase components in related organisms, such as the investigations of the αCTD in Mycobacterium smegmatis F₁-ATPase, where deletion of specific residues enhanced ATP hydrolysis activity .

What are the unique structural features of Anaeromyxobacter sp. ATP synthase subunit b that might reflect adaptation to its soil habitat?

Anaeromyxobacter species inhabit soil environments, particularly paddy soils, where they face fluctuating oxygen levels, varying pH, and changing nutrient availability . The ATP synthase b subunit likely incorporates structural adaptations that reflect these ecological challenges:

• Stability features: The b subunit may contain amino acid compositions that enhance stability under the variable conditions of soil environments. This could include salt bridges and hydrophobic interactions that maintain structural integrity across pH and temperature fluctuations.

• Interface with energy-conserving pathways: Given Anaeromyxobacter's capacity for both aerobic and anaerobic respiration, the b subunit might incorporate features that facilitate efficient energy coupling under varying redox conditions.

• Coordination with nitrogen fixation: As a diazotrophic bacterium , Anaeromyxobacter requires efficient energy production to support nitrogen fixation. The b subunit may contain adaptations that optimize ATP synthase performance during periods of high energy demand associated with nitrogenase activity.

• Regulatory interfaces: The b subunit might include binding sites for regulatory factors that modulate ATP synthase activity in response to environmental signals, similar to the regulatory elements identified in mycobacterial ATP synthases .

Comparative structural analysis with ATP synthase b subunits from organisms inhabiting different niches would help identify these adaptive features.

What purification strategies yield the highest activity and stability for recombinant Anaeromyxobacter sp. ATP synthase subunit b?

Purification of recombinant Anaeromyxobacter sp. ATP synthase subunit b requires careful consideration of its membrane protein nature. The following methodological approach is recommended based on successful purification of related ATP synthase components:

Throughout purification, it's critical to maintain a buffer system that mimics the physiological environment of Anaeromyxobacter, typically including:

• 50 mM Tris-HCl or HEPES (pH 7.5-8.0)

• 100-300 mM NaCl for ionic strength

• 5-10% glycerol as a stabilizing agent

• 0.05-0.1% DDM or equivalent detergent to maintain solubility

• 1-5 mM MgCl₂ to stabilize nucleotide-binding sites

This approach parallels successful purification strategies for ATP synthase components from other bacterial systems .

How can reconstitution experiments be designed to assess the functional integration of recombinant subunit b into the ATP synthase complex?

Reconstitution experiments provide critical insights into the functional role of recombinant Anaeromyxobacter sp. ATP synthase subunit b. A comprehensive experimental design would include:

• Preparation of components:

  • Purification of recombinant subunit b

  • Isolation of ATP synthase complex lacking subunit b

  • Preparation of liposomes with defined lipid composition

• Reconstitution protocol:

  • Sequential addition of proteins to detergent-destabilized liposomes

  • Detergent removal via bio-beads or dialysis

  • Assessment of protein orientation using protease accessibility assays

• Functional assays:

  • ATP synthesis measurement using luciferin/luciferase assay

  • ATP hydrolysis monitoring through phosphate release

  • Proton pumping assessment using pH-sensitive fluorescent dyes

  • Rotation assays using gold nanoparticles, similar to those performed with Mycobacterium smegmatis F₁-ATPase

• Comparative analysis:

  • Comparison with native ATP synthase complex

  • Assessment of the impact of mutations in subunit b

  • Evaluation of stability under various conditions

This experimental approach would provide insights into both the structural role of subunit b in complex assembly and its functional contribution to ATP synthesis activity.

What spectroscopic techniques are most informative for analyzing the structure and dynamics of Anaeromyxobacter sp. ATP synthase subunit b?

Several spectroscopic techniques offer valuable insights into the structure and dynamics of ATP synthase subunit b:

• Circular Dichroism (CD) Spectroscopy:

  • Provides secondary structure composition (α-helix, β-sheet content)

  • Monitors structural changes under varying conditions (pH, temperature)

  • Assesses thermal stability through temperature-dependent measurements

• Fluorescence Spectroscopy:

  • Intrinsic tryptophan fluorescence reveals tertiary structure changes

  • Site-specific labeling with fluorescent probes enables dynamic studies

  • Fluorescence resonance energy transfer (FRET) measures distances between labeled sites

• Nuclear Magnetic Resonance (NMR) Spectroscopy:

  • For isolated domains: high-resolution structural information

  • Chemical shift perturbation experiments identify binding interfaces

  • Relaxation measurements provide insights into protein dynamics

• Electron Paramagnetic Resonance (EPR) Spectroscopy:

  • Site-directed spin labeling coupled with EPR measures distances and orientations

  • Double electron-electron resonance (DEER) determines long-range distances

  • Continuous wave EPR monitors local environmental changes

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS):

  • Maps solvent-accessible regions and secondary structure elements

  • Identifies conformational changes upon binding to other subunits

  • Requires minimal sample amount compared to other structural techniques

These spectroscopic approaches complement the structural insights gained from cryo-EM studies of ATP synthases from related organisms .

How can structural knowledge of Anaeromyxobacter sp. ATP synthase subunit b inform the development of inhibitors for antimicrobial applications?

Understanding the structure of Anaeromyxobacter sp. ATP synthase subunit b could facilitate the development of targeted inhibitors through the following research strategy:

• Identification of unique structural features:

  • Comparative structural analysis with human ATP synthase

  • Mapping of species-specific regions that could serve as selective targets

  • Identification of critical residues for subunit-subunit interactions

• Rational inhibitor design approach:

  • Structure-based virtual screening against identified binding pockets

  • Fragment-based drug discovery focusing on interface regions

  • Peptide mimetics targeting species-specific interaction surfaces

• Validation methodology:

  • Binding assays using surface plasmon resonance or isothermal titration calorimetry

  • Functional inhibition assessment through ATP synthesis/hydrolysis assays

  • Structural confirmation of binding mode through co-crystallization or cryo-EM

This approach parallels successful targeting strategies for mycobacterial ATP synthases, where species-specific elements like the αCTD and γ-loop have been identified as attractive targets for inhibitor development . Similar species-specific elements in Anaeromyxobacter ATP synthase could serve as targets for selective inhibitors.

What insights can comparative studies between Anaeromyxobacter sp. ATP synthase and related bacterial ATP synthases provide about evolutionary adaptations?

Comparative studies of ATP synthases across bacterial species can reveal evolutionary adaptations that reflect diverse ecological niches and metabolic strategies:

• Structural comparison framework:

  • Alignment of primary sequences from diverse bacterial sources

  • Comparison of tertiary structures, focusing on peripheral stalk components

  • Analysis of interface regions between subunits

• Functional correlations:

  • Assessment of ATP hydrolysis regulation mechanisms across species

  • Comparison of energy coupling efficiency under various conditions

  • Evaluation of stability in different environmental contexts

• Ecological context analysis:

  • Correlation of structural features with habitat characteristics

  • Comparison between soil-dwelling bacteria and those from other environments

  • Examination of adaptations in diazotrophic versus non-diazotrophic bacteria

The mycobacterial ATP synthase exhibits unique features like the extended C-terminal domain (αCTD) of subunit α, which serves as the main element for self-inhibition of ATP hydrolysis . Similar distinctive elements in Anaeromyxobacter ATP synthase could represent evolutionary adaptations to its specific ecological niche as a soil-dwelling, facultatively aerobic bacterium with nitrogen-fixing capabilities .

How does the function of ATP synthase in Anaeromyxobacter sp. relate to its nitrogen fixation capabilities?

Anaeromyxobacter species possess both ATP synthase and nitrogen fixation machinery, creating an important energetic relationship that can be explored through the following research approaches:

• Energetic coupling analysis:

  • Measurement of ATP synthesis rates under nitrogen-fixing versus non-fixing conditions

  • Assessment of ATP consumption by nitrogenase versus other cellular processes

  • Quantification of energy allocation during simultaneous nitrogen fixation and growth

• Regulatory network investigation:

  • Analysis of transcriptional coordination between ATP synthase and nitrogenase genes

  • Examination of post-translational regulation mechanisms affecting both systems

  • Investigation of metabolic signaling pathways that coordinate energy production and nitrogen fixation

• Structural adaptation assessment:

  • Evaluation of ATP synthase features that may support high energy demand during nitrogen fixation

  • Comparison with ATP synthases from non-diazotrophic relatives

  • Identification of potential interaction sites between energy production and nitrogen fixation machinery

Anaeromyxobacter contains a compact nif gene cluster (nifHDKENX-fdxN) responsible for nitrogen fixation , which creates significant energy demands. The ATP synthase must supply sufficient ATP to support both nitrogen fixation and normal cellular functions, suggesting potential adaptations in its structure and regulation compared to non-diazotrophic bacteria.

What experimental approaches can determine if structural features found in mycobacterial ATP synthases, such as the γ-loop, are also present in Anaeromyxobacter sp. ATP synthase?

To investigate whether structural features like the mycobacterial γ-loop exist in Anaeromyxobacter sp. ATP synthase, the following experimental approaches are recommended:

• Genomic and bioinformatic analysis:

  • Sequence alignment of ATP synthase γ subunit across species

  • Secondary structure prediction to identify potential loop regions

  • Homology modeling based on existing ATP synthase structures

• Structural biology approaches:

  • Cryo-EM analysis of purified Anaeromyxobacter ATP synthase complex

  • X-ray crystallography of isolated γ subunit

  • Hydrogen-deuterium exchange mass spectrometry to map flexible regions

• Functional validation methods:

  • Generation of deletion mutants targeting predicted loop regions

  • Measurement of ATP synthesis/hydrolysis activities in mutants

  • Rotational studies using gold nanoparticles to assess mechanistic impacts

• Cross-species hybrid experiments:

  • Creation of chimeric proteins with γ subunit components from different species

  • Assessment of functional compatibility and activity in reconstituted systems

  • Evaluation of species-specific regulatory mechanisms

This multi-faceted approach would reveal whether Anaeromyxobacter ATP synthase contains structural elements similar to the mycobacterial γ-loop, which has been identified as essential for catalysis and a potential target for species-specific inhibitors like GaMF1 .

What are the most promising research directions for understanding the role of Anaeromyxobacter sp. ATP synthase in soil ecosystem functions?

Future research on Anaeromyxobacter sp. ATP synthase should focus on its ecological significance and potential applications:

• Ecosystem energetics:

  • Investigation of ATP synthase activity under varying soil conditions

  • Assessment of energy conservation efficiency across redox gradients

  • Quantification of contribution to soil microbial community energy flow

• Biogeochemical cycling:

  • Exploration of linkages between ATP synthesis and nitrogen fixation in soil environments

  • Investigation of ATP synthase activity during metal reduction processes

  • Analysis of energy contribution to carbon cycling in anaerobic soil microsites

• Climate change adaptation:

  • Examination of ATP synthase performance under altered temperature regimes

  • Assessment of efficiency under flooding conditions (relevant to paddy soils)

  • Investigation of drought response mechanisms related to energy conservation

• Biotechnological applications:

  • Evaluation of potential for engineered bioremediation applications

  • Assessment of suitability for bioenergy production systems

  • Investigation as a model for designing robust energy-conserving systems

These research directions would build upon current understanding of Anaeromyxobacter as an important soil bacterium with diazotrophic capabilities and expand knowledge of how its energy conservation systems support ecological functions.

How can advances in structural biology techniques improve our understanding of the complete Anaeromyxobacter sp. ATP synthase complex?

Recent advances in structural biology offer unprecedented opportunities to elucidate the structure and dynamics of the complete Anaeromyxobacter sp. ATP synthase:

• Cryo-electron microscopy advancements:

  • Single-particle analysis at near-atomic resolution

  • Time-resolved cryo-EM to capture different conformational states

  • Cryo-electron tomography to visualize ATP synthase in cellular context

• Integrative structural biology approaches:

  • Combination of cryo-EM with mass spectrometry for subunit mapping

  • Integration of molecular dynamics simulations with experimental data

  • Cross-linking mass spectrometry to define subunit interfaces

• Advanced spectroscopic methods:

  • Site-directed spin labeling EPR for dynamics studies

  • Single-molecule FRET to monitor conformational changes during catalysis

  • Solid-state NMR to analyze membrane-embedded portions

• Computational approaches:

  • Molecular dynamics simulations of the complete complex

  • Coarse-grained modeling to capture large-scale conformational changes

  • Machine learning approaches for structure prediction from sequence